The enhancement by surfactants of hexadecane degradation by Pseudomonas aeruginosa varies with substrate availability

Abstract

The rhamnolipid biosurfactant produced by Pseudomonas aeruginosa influences various processes related to hydrocarbon degradation. However, degradation can only be enhanced by the surfactant when it stimulates a process that is rate limiting under the applied conditions. Therefore we determined how rhamnolipid influences hexadecane degradation by P. aeruginosa UG2 under conditions differing in hexadecane availability. The rate of hexadecane degradation in shake flask cultures was lower for hexadecane entrapped in a matrix with 6 nm pores (silica 60) or in quartz sand than for hexadecane immobilized in matrices with pore sizes larger than 300 nm or for hexadecane present as a separate liquid phase. This indicates that the availability of hexadecane decreased with decreasing pore size under these conditions. The rate-limiting step for hexadecane entrapped in silica 60 was the mass transfer of substrate from the matrix to the bulk liquid phase, whereas for hexadecane present as a second liquid phase it was the uptake of the substrate by the cells. Hexadecane degradation in batch incubations was accelerated by the addition of rhamnolipid or other surfactants in all experiments except in those where hexadecane was entrapped in silica 60, indicating that the surfactants stimulated uptake of hexadecane by the cells. Since rhamnolipid stimulated the degradation rate in batch experiments to a greater extent than any of the other 14 surfactants tested, hexadecane uptake was apparently more enhanced by rhamnolipid than by the other surfactants. Although rhamnolipid did not stimulate the release of hexadecane from silica 60 under conditions of intense agitation, it significantly enhanced this rate during column experiments in the absence of strain UG2. The results demonstrate that rhamnolipid enhances degradation by stimulating release of entrapped substrate in column studies under conditions of low agitation and by stimulating uptake of substrate by the cells, especially when degradation is not limited by release of substrate from the matrices.

Introduction

The use of surfactants to overcome bioavailability-associated limitations during soil remediation has attracted considerable attention (Miller, 1995, Volkering et al., 1998). Positive effects of surfactants may result from a stimulation of dissolution or desorption rates (Volkering et al., 1995, Grimberg et al., 1996, Mulder et al., 1998, Willumsen and Arvin, 1999) or from surfactant-mediated dispersion, solubilization, or emulsification of poorly soluble substrates (Aronstein et al., 1991, Tiehm, 1994, Miller, 1995, Volkering et al., 1998). Negative effects may also occur, however, for example because a surfactant may be toxic or due to preferential biodegradation of surfactants (Miller, 1995, Volkering et al., 1998). Furthermore, surfactants may reduce attachment of cells to substrates that are present as a separate phase, which can decrease degradation rates if attachment is needed for uptake (Churchill and Churchill, 1997, Herman et al., 1997b). Despite these general trends, the effect of surfactants and biosurfactants on the biodegradation of organic compounds is poorly predictable.

The rhamnolipid biosurfactant produced by P. aeruginosa can stimulate the biodegradation of long-chain alkanes by this strain, both when these compounds are present as a separate liquid phase (Itoh and Suzuki, 1972, Nakahara et al., 1981, Koch et al., 1991, Zhang and Miller, 1992) and when the substrate is present as a residual non-aqueous phase in soil (Herman et al., 1997b). Although substrate dispersion may be required, the stimulation of dispersion by rhamnolipid was not reflected by a proportional increase of the degradation rate (Zhang and Miller, 1994, Zhang and Miller, 1995). Therefore, it was concluded that rhamnolipid stimulated degradation both by enhancing dispersion of substrate and by increasing cell surface hydrophobicity (Zhang and Miller, 1994, Zhang and Miller, 1995, Herman et al., 1997b). In contrast, inhibition of octadecane degradation by rhamnolipid also occurred, which may have resulted from the interference of cell-hydrocarbon interaction by the surfactant (Zhang and Miller, 1994). Recently, it was shown that rhamnolipid extracts lipopolysaccharides (LPS) from cells of Pseudomonas, thereby increasing the hydrophobicity of the cell surface and promoting attachment of the cells to hydrocarbon droplets (Al-Tahhan et al., 2000). It was suggested that this greater attachment stimulates hexadecane degradation (Al-Tahhan et al., 2000). When hexadecane was present as a residual liquid in soil, rhamnolipid increased degradation by some organisms but inhibited degradation by other strains (Herman et al., 1997a, Herman et al., 1997b). The stimulating effect of rhamnolipid was attributed to enhanced transport of substrate to the bacteria and the inhibitory effect to rhamnolipid-induced flocculation of the cells. It is known that the stimulation of many P. aeruginosa strains is more pronounced for rhamnolipid than for other surfactants (Itoh and Suzuki, 1972, Nakahara et al., 1981), but the reason behind this specificity is unknown.

It has become clear that rhamnolipid stimulates different processes related to the degradation of organic substrates. Since several steps are involved in the degradation of a poorly soluble compound, a biosurfactant will only enhance degradation when the step that is stimulated is rate limiting. The step that is rate limiting may differ between different conditions. Therefore, the extent to which rhamnolipid enhances the degradation rate or even the way how the surfactant influences this rate probably depends on the form in which the substrate is present.

The goal of this work was to obtain insight into how rhamnolipid stimulates degradation of hexadecane by P. aeruginosa UG2 under conditions differing in the availability of the substrate. The availability varied by using hexadecane that occurs as a separate liquid phase and hexadecane present in different matrices varying in pore size. Furthermore, different hydrodynamic conditions were employed, i.e. conditions of high agitation as present during shake flask experiments and conditions of low agitation as observed during continuous flow operation of columns packed with the contaminated matrices. The approach was to determine which step in the degradation was rate limiting under these different conditions and to subsequently investigate whether rhamnolipid stimulated that step. In the analysis we discerned three basic steps: mass transfer of inaccessible substrate from the matrix to the bulk phase, solubilization or emulsification of the substrate to a form that can be taken up by the cells, and uptake of (solubilized or emulsified) substrate by the cells. Hexadecane was used as the model substrate, since it is easily degraded by strain UG2 and has an extremely low aqueous solubility. It is anticipated that the results with this model substrate are relevant for rhamnolipid-enhanced degradation of all liquid hydrophobic compounds that are a substrate for strain UG2. To determine which step in the degradation of hexadecane was rate limiting, degradation studies in shake flasks were conducted. Subsequently, it was determined whether the degradation could be stimulated by surfactants. To better understand which step in the degradation by P. aeruginosa was stimulated by surfactants, the effect of rhamnolipid on degradation and emulsification was compared to the effect of several other surfactants. Column studies were conducted in the absence of cells to determine whether rhamnolipid enhanced mass transfer of hexadecane from the matrix to the aqueous under conditions of low agitation.